Over the past grant cycle, general rules for calmodulin (CaM) regulation of the family of Ca channels were discerned. CaM has two lobes, each with two Ca2+ binding sites. A first general aspect was that each lobe can autonomously trigger a form of channel regulation. Secondly, whenever the C-terminal lobe of CaM (C-lobe) triggers channel regulation, there is preferential responsiveness to local Ca2+ signals. Conversely, wherever the N-terminal lobe (N-lobe) initiates regulation, there is selectivity for global Ca2+ signals. Thirdly, there is reason to expect that this rule of operation generalizes beyond Ca channels, to many complexes in which CaM is preassociated with target molecules. Because CaM regulation of Ca channels (and other signaling molecules) is crucial for normal neuroprocessing, and likely important for therapeutics relating to pain, psychosis, and cardiac arrhythmogenesis, answering how these general rules occur is the overarching thrust for the next cycle of research. Three aims will address this overall theme. 1. To develop and perform elementary tests of a kinetic Ca2+ decoding mechanism for the CaM/Ca channel complex. This aim formulates a 'kinetic Ca2+ decoding'theory of how the CaM decoding occurs, and devises novel Voltage-block'experiments to enable elementary tests of this theory. 2. To engineer the local/global Ca2+ preference of CaM/Ca channel regulation, as a higher-order test of the kinetic Ca2+ decoding theory, and as means to glean design principles for developing novel channel modulators. A principal prediction of the kinetic Ca2+ decoding theory is that the local/global Ca2+ preference of channel regulation reflects competition between channel affinities for the Ca2+-bound and Ca2+-free forms of a lobe of CaM. Aim 2 will alter these affinities and check for the predicted changes in Ca2+ preference. Aim 2 will also explore whether these modifications can inform the design of drug compounds that modulate channel regulation in new ways. 3. To experimentally determine Ca2+ concentrations and diffusion within the nanometers of the Ca channel. Crucial to the next phase of progress is the direct measurements of local and global Ca2+ concentration signals, and Ca2+ diffusion, in the actual channel 'nanodomain'environment. Fusions of a genetically-encoded Ca2+ sensor (TNL-15) to channels, combined with TIRF microscopy promise to reveal these long sought-after entities. These aims promise bold progress, with basic and applied ramifications.